CN101792806A - Detection method of single-stranded micro ribonucleic acid - Google Patents

Abstract

The invention relates to a detection method of single-stranded micro ribonucleic acid (miRNA), belonging to the technical field of biological detection. Cells are used or RNA (containing miRNA) is organized, wherein a polyadenylic acid tail is added to the 3' terminal of miRNA by poly (a) polymerase, then reverse transcription primers are used for reverse transcription to obtain single-stranded deoxyribonucleic acid (ssDNA) complementary to the RNA, finally the miRNA related sequences and partial complementary sequences of the reverse transcription primers are respectively used as the upperstream and downstream primers for miRNA detection and a specific detection system (containing SYBR Green I fluorescent dye) is used for detecting miRNA. The method has the advantages of simpleness, convenience, rapidness and suitability for detection of a large number of samples. The method can be used for expression analysis, cloning research and clinical sample test of miRNA.

Description

A kind of detection method of single-stranded micro ribonucleic acid

Technical field

The present invention relates to a kind of detection method of single-stranded micro ribonucleic acid, belong to technical field of biological.

Background technology

Single-stranded micro ribonucleic acid (hereinafter to be referred as miRNA) is the ribonucleic acid molecule that a class has 21-23 mononucleotide length, by the higher eucaryote genome encoding, miRNA is by guiding silencing complex (RISC) degraded messenger RNA(mRNA) or hinder its translation with the base pairing of target gene messenger RNA(mRNA).MiRNAs is quite conservative in spore, the miRNA that finds in plant, animal and fungi only expresses at specific tissue and etap, miRNA tissue specificity and sequential, the function specificity of decision tissue and cell shows that miRNA plays multiple effect in the regulate process of cell g and D process.The miRNA sequence has certain conservative property in different biomass cellss, the function of miRNA is some primary processes that participate in life, as the cell proliferation in the growth course, necrocytosis, stress reaction and metabolism of fat etc.Nearest discovers, miRNA expresses relevant with multiple cancer, and about 50% miRNAs that obtains explaining is positioned the fragile site relevant with tumour on genome.This explanation miRNAs plays crucial effects in the tumour generating process, these miRNAs roles are similar to the function of cancer suppressor gene and oncogene.These results of study show that all miRNA may play an important role in cell carcinogenesis.Therefore, set up that a kind of to operate the detection method fast and convenient, that expense is cheap significant to the functional study of miRNA.Because the miRNA molecule is less, the difficult point of detection technique is higher than the detection of all the other molecules.Sophisticated miRNA generally has only twenties bases, usually the difference of having only one or two base between the consanguinity miRNA, and the expression level of the overwhelming majority in cell is all lower, these characteristics are given traditional detection method such as northern hybridization, and conventional meanses such as chip detection have brought great difficulty and challenge.

Summary of the invention

The objective of the invention is to propose a kind of detection method of single-stranded micro ribonucleic acid, set up a kind of easy, practical method that is used to detect the miRNA molecule, with expression analysis, clone's research and the clinical samples check that is used for miRNA.

The detection method of the single-stranded micro ribonucleic acid that the present invention proposes may further comprise the steps:

(1) Yeast Nucleic Acid that will comprise single-stranded micro ribonucleic acid joins in the poly A polymerase reactive system, 37 ℃ of reactions 50-70 minute, obtain reaction solution, the volume of described poly A polymerase reactive system is 20 microlitres, the mass volume ratio that Yeast Nucleic Acid adds is 2 nanograms～200 nanograms/microlitre, the consisting of of poly A polymerase reactive system: poly A polymerase is 2 units, Yeast Nucleic Acid is 20 nanograms～2 micrograms, the pH value be 8.0 Tri(Hydroxymethyl) Amino Methane Hydrochloride 100 mmoles/liter, sodium-chlor 200 mmoles/liter, ethylenediamine tetraacetic acid (EDTA) 80 mmoles/liter and Triphosaden be 50 mmoles/liter;

(2) from the reaction solution of step (1), take out 2 microlitres, join in the reverse transcription reaction system, 37 ℃ of reactions 50-70 minute, obtain reaction solution, the volume of reverse transcription reaction system is 20 microlitres, the adding volume ratio of the reaction solution of step (1) is 1: 10, and the reverse transcription reaction system consists of: reaction solution 2 microlitres of step (1), reverse transcriptase primer 150 nmoles/liter, the pH value be 8.8 Tri(Hydroxymethyl) Amino Methane Hydrochloride 150 mmoles/liter, Repone K 500 mmoles/liter, the triphosphoric acid dezyribonucleoside is 100 micromoles per liter, ribonuclease inhibitor is that 20 units and Quant reversed transcriptive enzyme are 2 units;

(3) from the reaction solution of step (2), take out 2 microlitres, join in the system of polymerase chain reaction, response procedures is 40 thermal cyclings, the condition of each thermal cycling is 94 ℃ of insulations 20 seconds, 60 ℃ are incubated 30 seconds, 72 ℃ are incubated 30 seconds, the adding volume ratio of step (2) reaction solution is 1: 10, and the polymerase chain reaction system consists of: reaction solution 2 microlitres of step (2), deoxyribonucleic acid polymerase mixed solution 10 microlitres, upstream primer volumetric molar concentration be 200 nmoles/liter and universal downstream primer volumetric molar concentration be 200 nmoles/liter.

The detection method of the single-stranded micro ribonucleic acid that the present invention proposes, its advantage are to detect easy, quick, saving.The inventive method is based on the miRNA fluorescent quantitation detection system of SYBR Green I dye method, can be used for from the single stranded deoxyribonucleic acid of a building-up reactions, detecting multiple miRNA, not only reduce the detection error, saved test sample, also realized the high-throughput that detects simultaneously.And the inventive method has the quite good detecting specificity, can differentiate the miRNA of single base difference; The detection sensitivity height can detect the miRNA of specifically expressing in being low to moderate total Yeast Nucleic Acid of nanogram level.

Description of drawings

Fig. 1 is the amplification curve that single-stranded micro ribonucleic acid mir-24 and mir-122 fluorescent quantitation detect.

Fig. 2 is the solubility curve that single-stranded micro ribonucleic acid mir-24 and mir-122 fluorescent quantitation detect.

Embodiment

The detection method of the single-stranded micro ribonucleic acid that the present invention proposes may further comprise the steps:

(1) Yeast Nucleic Acid that will comprise single-stranded micro ribonucleic acid joins in the poly A polymerase reactive system, 37 ℃ of reactions 50-70 minute, obtain reaction solution, the volume of described poly A polymerase reactive system is 20 microlitres, the mass volume ratio that Yeast Nucleic Acid adds is 2 nanograms～200 nanograms/microlitre, the consisting of of poly A polymerase reactive system: poly A polymerase is 2 units, Yeast Nucleic Acid is 20 nanograms～2 micrograms, the pH value be 8.0 Tri(Hydroxymethyl) Amino Methane Hydrochloride 100 mmoles/liter, sodium-chlor 200 mmoles/liter, ethylenediamine tetraacetic acid (EDTA) 80 mmoles/liter and Triphosaden be 50 mmoles/liter;

(2) from the reaction solution of step (1), take out 2 microlitres, join in the reverse transcription reaction system, 37 ℃ of reactions 50-70 minute, obtain reaction solution, the volume of reverse transcription reaction system is 20 microlitres, the adding volume ratio of the reaction solution of step (1) is 1: 10, and the reverse transcription reaction system consists of: reaction solution 2 microlitres of step (1), reverse transcriptase primer 150 nmoles/liter, the pH value be 8.8 Tri(Hydroxymethyl) Amino Methane Hydrochloride 150 mmoles/liter, Repone K 500 mmoles/liter, the triphosphoric acid dezyribonucleoside is 100 micromoles per liter, ribonuclease inhibitor is that 20 units and Quant reversed transcriptive enzyme are 2 units;

(3) from the reaction solution of step (2), take out 2 microlitres, join in the system of polymerase chain reaction, response procedures is 40 thermal cyclings, the condition of each thermal cycling is 94 ℃ of insulations 20 seconds, 60 ℃ are incubated 30 seconds, 72 ℃ are incubated 30 seconds, the adding volume ratio of step (2) reaction solution is 1: 10, and the polymerase chain reaction system consists of: reaction solution 2 microlitres of step (2), deoxyribonucleic acid polymerase mixed solution 10 microlitres, upstream primer volumetric molar concentration be 200 nmoles/liter and universal downstream primer volumetric molar concentration be 200 nmoles/liter.

The present invention relates to a kind of detection method of single-stranded micro ribonucleic acid, belong to technical field of biological.Use cell or tissue Yeast Nucleic Acid (containing miRNA), miRNA wherein adds the polyadenylic acid tail through poly A polymerase at its 3 ' end, carry out reverse transcription by reverse transcriptase primer again and obtain the complementary single stranded deoxyribonucleic acid of Yeast Nucleic Acid, the last upstream and downstream primer that detects as miRNA respectively with the part complementary sequence of miRNA correlated series and reverse transcriptase primer uses specific detection system (containing SYBR Green I fluorescence dye) to detect miRNA.

In the inventive method, used Quant reversed transcriptive enzyme and deoxyribonucleic acid polymerase mixed solution are produced by TIANGEN Biotech (Beijing) Co., Ltd..

The upstream primer, the universal downstream primer that use in the reverse transcriptase primer that uses in the step in the aforesaid method (2), the step (3), synthetic by the U.S. handsome invitrogen company.

Below introduce the embodiment of the inventive method: detect single-stranded micro ribonucleic acid mir-24 and single-stranded micro ribonucleic acid mir-122 in the mouse liver tissue.

1, poly A polymerase reaction

(1) comprise the preparation of the Yeast Nucleic Acid of single-stranded micro ribonucleic acid:

Take by weighing the mouse liver tissue of 50mg, with the total Yeast Nucleic Acid in the miRNA extraction test kit extraction mouse liver cell of TIANGEN Biotech (Beijing) Co., Ltd., the total Yeast Nucleic Acid that obtains is measured through light absorption value and is obtained its concentration.

(2) preparation poly A polymerase reaction mixture 20 microlitres, composed as follows: Yeast Nucleic Acid is 2 micrograms, poly A polymerase is 2U, the pH value be 8.0 Tri(Hydroxymethyl) Amino Methane Hydrochloride 100 mmoles/liter, sodium-chlor 200 mmoles/liter, ethylenediamine tetraacetic acid (EDTA) 80 mmoles/liter and Triphosaden be 50 mmoles/liter.The reaction solution of the above-mentioned preparation of mixing reacted 50-70 minute at 37 ℃ gently, and the 3 ' end of miRNA closes adenylic acid (AMP) tail structure for poly in the reaction end afterreaction liquid.The reaction solution of gained can directly carry out the downstream experiment, also can place-20 ℃ of of short duration preservations, and suggestion is deposited in-80 ℃ as the need prolonged preservation.

2, reverse transcription reaction

(1) sequence of reverse transcriptase primer:

5’-GCGCAATGCATCGCATAGCAACTGTGCATGACAGTG(T)22(A，G，C)-3’

(2) preparation reverse transcription reaction mixed solution 20 microlitres, composed as follows: reaction solution 2 microlitres of step 1 (2), reverse transcriptase primer 150 nmoles/liter, the pH value be 8.8 Tri(Hydroxymethyl) Amino Methane Hydrochloride 150 mmoles/liter, Repone K 500 mmoles/liter, the triphosphoric acid dezyribonucleoside is that 100 micromoles per liter, ribonuclease inhibitor are that 20 units and Quant reversed transcriptive enzyme are 2 units.The reaction solution of the above-mentioned preparation of mixing 37 ℃ of reactions 50-70 minute, contains corresponding single stranded deoxyribonucleic acid in the reaction end afterreaction liquid gently, is positioned over-20 ℃ of preservations.

3, polymerase chain reaction

(1) primer sequence:

Universal downstream: 5 '-GCGCAATGCATCGCATAGCAACTGT-3 '

Mir-24 upstream primer sequence: 5 '-TGGCTCAGTTCAGCAGGAACAG-3 '

Mir-122 upstream primer sequence: 5 '-TGGAGTGTGACAATGGTGTTTGT-3 '

(2) preparation mixed liquid of polymerase chair reaction 20 microlitres, composed as follows: reaction solution 2 microlitres of step 2 (2), deoxyribonucleic acid polymerase mixed solution 10 microlitres of TIANGEN Biotech (Beijing) Co., Ltd., the upstream primer volumetric molar concentration be 200 nmoles/liter, universal downstream primer volumetric molar concentration be 200 nmoles/liter.Response procedures is 40 thermal cyclings, and the condition of each thermal cycling is 94 ℃ of insulations 20 seconds, and 60 ℃ are incubated 30 seconds, and 72 ℃ are incubated 30 seconds, and reaction can detect corresponding miRNA by the fluorescent quantitation detector after finishing.Fig. 1 is the amplification curve that single-stranded micro ribonucleic acid mir-24 and mir-122 fluorescent quantitation detect in the Figure of description; Fig. 2 is the solubility curve that single-stranded micro ribonucleic acid mir-24 and mir-122 fluorescent quantitation detect.

Claims (1)

1. the detection method of a single-stranded micro ribonucleic acid is characterized in that this method may further comprise the steps:

(1) Yeast Nucleic Acid that will comprise single-stranded micro ribonucleic acid joins in the poly A polymerase reactive system, 37 ℃ of reactions 50-70 minute, obtain reaction solution, the volume of described poly A polymerase reactive system is 20 microlitres, the mass volume ratio that Yeast Nucleic Acid adds is 2 nanograms～200 nanograms/microlitre, the consisting of of poly A polymerase reactive system: poly A polymerase is 2 units, Yeast Nucleic Acid is 20 nanograms～2 micrograms, the pH value be 8.0 Tri(Hydroxymethyl) Amino Methane Hydrochloride 100 mmoles/liter, sodium-chlor 200 mmoles/liter, ethylenediamine tetraacetic acid (EDTA) 80 mmoles/liter and Triphosaden be 50 mmoles/liter;

(2) from the reaction solution of step (1), take out 2 microlitres, join in the reverse transcription reaction system, 37 ℃ of reactions 50-70 minute, obtain reaction solution, the volume of reverse transcription reaction system is 20 microlitres, the adding volume ratio of the reaction solution of step (1) is 1: 10, and the reverse transcription reaction system consists of: reaction solution 2 microlitres of step (1), reverse transcriptase primer 150 nmoles/liter, the pH value be 8.8 Tri(Hydroxymethyl) Amino Methane Hydrochloride 150 mmoles/liter, Repone K 500 mmoles/liter, the triphosphoric acid dezyribonucleoside is 100 micromoles per liter, ribonuclease inhibitor is that 20 units and Quant reversed transcriptive enzyme are 2 units;

(3) from the reaction solution of step (2), take out 2 microlitres, join in the system of polymerase chain reaction, response procedures is 40 thermal cyclings, the condition of each thermal cycling is 94 ℃ of insulations 20 seconds, 60 ℃ are incubated 30 seconds, 72 ℃ are incubated 30 seconds, the adding volume ratio of step (2) reaction solution is 1: 10, and the polymerase chain reaction system consists of: reaction solution 2 microlitres of step (2), deoxyribonucleic acid polymerase mixed solution 10 microlitres, upstream primer volumetric molar concentration be 200 nmoles/liter and universal downstream primer volumetric molar concentration be 200 nmoles/liter.

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